Binding of epidermal growth factor (EGF) to its receptor activates the receptor's tyrosine kinase activity. Subsequently. the stimulated receptor phosphorylates itself and other intracellular proteins. These intracellular proteins are collectively termed EGF receptor tyrosine kinase substrates. A method was developed to isolate these substrates and several cDNA clones were obtained. They were designated eps for EGF receptor pathway substrate. from an NIH/3T3 cell cDNA library. One of these clones, eps8, encodes a protein of 97 kDa. It was shown that eps8 was phosphorylated by the activated EGF receptor tyrosine kinase as well as by other tyrosine kinases. eps8 binds to the EGF receptor directly. despite the absence of a SH2 domain. It was further determined that the regions of eps8 are important for its interaction with the EGF receptor. Overexpression of recombinant eps8 in fibroblasts or hematopoietic cells increases the cells response and sensitivity to the mitogenic action of EGF. Therefore, eps8 is likely involved in the transduction of EGF signals. Sequence analysis revealed a SH3 domain in eps8. A 65 kDa protein was identified that binds to the SH3 domain of eps8 and showed that this protein is mainly distributed in the nuclear fraction of the cell.